1. Field
The invention is in the field of biochemical methods of testing for the presence of phospholipases, particularly phospholipase C, including methods for the preparation of aqueous based dispersions of phospholipids.
2. State of the Art
It is often desirable to test for the presence of phospholipases, particularly phospholipase C. Phospholipase C is an enzyme capable of reacting with and breaking down lecithin. This adverse reaction ultimately leads to cell breakdown and tissue destruction.
The presence of phospholipase C with Clostridium, Staphylococcus, or Pseudomonas bacteria indicates the pathogenic species of such bacteria, while absence of phospholipase C indicates the non-pathogenic species. Absence of phospholipase C with Bacillus bacteria indicates the pathogenic species of that bacteria.
Presently there are two methods of testing for the presence of or, if present, for activity of the phospholipase C enzyme. One method uses either purified lecithin or egg yolks (egg yolks contain lecithin) in a saline solution to which the material to be tested for the presence of phospholipase enzyme is added. The phospholipase enzyme splits the lecithin in the saline solution into an acid soluable phosphate. The amount of phosphate in the solution is then measured by usual means such as by a spectrophotometer, the amount of phosphate being an indication of the presence and activity of the phopholipase. Since this test measures phosphate, it is limited in its sensitivity to the sensitivity of the phosphate test.
Also, in order to obtain a suspension of lecithin in an aqueous based saline solution when using purified lecithin, the lecithin-saline solution must be sonicated (bombarded by sound waves) to break the lecithin down into micelles. The micelles then form a suspension in the solution. However, the micelles formed by sonication are not uniform, and, thus, the suspension in the saline solution is not uniform. This makes it extremely difficult to get consistant results. Further, because lecithin breaks down very rapidly at elevated temperatures, the purified lecithin cannot be sterilized. This means that a sterilized lecithin-saline solution cannot be obtained. Thus, when purified lecithin is used, the test cannot have clinical application in instances in which a sterile solution is necessary. It is also difficult to obtain the same results with phospholipase enzymes from different genera when using purified lecithin derived from different lecithin sources, e.g. purified lecithin derived from egg yolks or soybeans.
Egg yolks, when used, are difficult to handle because sterile precautions must be observed and shelf life of the egg yolks or egg yolk-saline solution is limited to only about a week. Further, the egg yolks give inconsistant results from test to test, and although presence of phospholipase enzyme may be determined, it is difficult to standardize the tests to obtain quantitative results.
The second test currently in use utilizes egg yolk in an agar medium. The material being tested for presence or activity of phospholipase enzyme is introduced into wells, i.e. depressions, formed in the agar. The phospholipase enzyme breaks down the lecithin in the agar. This breaking down or hydrolysis of the lecithin causes discoloration (darkening) of the agar medium. The amount and extent of hydrolysis extending from the well indicates directly the presence and activity of the phospholipase enzyme. The advantage of this test is that the phospholipase activity is measured directly, rather than indirectly as by measuring phosphate content in the test previously described, and is thus much more sensitive. The same disadvantage of egg yolk handling and test inconsistencies are present with this test using egg yolks as described in the previous test. In addition, the egg yolk-agar is non-specific, i.e. positive results can be obtained with enzymes other than phospholipase because of reaction of components of the egg yolk other than lecithin.
The agar test, although having advantages over the saline solution test, has not utilized purified lecithin because no method has yet been found to suspend the lecithin in the agar medium and maintain a uniform suspension during the time the medium is hardening. A uniform suspension is necessary for reproduceable results. The use of purified lecithin would be advantageous because of elimination of the other components of egg yolk, which can cause the positive reaction to occur without the phospholipase being present, and because a known uniform concentration of lecithin can be used from test to test.